The present invention relates to the field of food manufacturing, in particular to the preparation of improved bakery products and other farinaceous food products. Specifically, the invention concerns the use of glycerol oxidase as a dough strengthening agent and improvement of the quality of baked and dried products made from such improved doughs. There is also provided a method of improving the properties of doughs and baked product by combined use of glycerol oxidase and a lipase.
The xe2x80x9cstrengthxe2x80x9d or xe2x80x9cweaknessxe2x80x9d of doughs is an important aspect of making farinaceous finished products from doughs, including baking. The xe2x80x9cstrengthxe2x80x9d or xe2x80x9cweaknessxe2x80x9d of a dough is primarily determined by its content of protein and in particular the content and the quality of the gluten protein is an important factor in that respect. Flours with a low protein content are generally characterized as xe2x80x9cweakxe2x80x9d. Thus, the cohesive, extensible, rubbery mass which is formed by mixing water and weak flour will usually be highly extensible when subjected to stress, but it will not return to its original dimensions when the stress is removed.
Flours with a high protein content are generally characterized as xe2x80x9cstrongxe2x80x9d flours and the mass formed by mixing such a flour and water will be less extensible than the mass formed from a weak flour, and stress which is applied during mixing will be restored without breakdown to a greater extent than is the case with a dough mass formed from a weak flour. Strong flour is generally preferred in most baking contexts because of the superior rheological and handling properties of the dough and the superior form and texture qualities of the finished baked or dried products made from the strong flour dough. Doughs made from strong flours are generally more stable. Stability of a dough is one of the most important characteristics of flour doughs. Within the bakery and milling industries it is known to use dough xe2x80x9cconditionersxe2x80x9d to strengthen the dough to increase its stability and strength. Such dough conditioners are normally non-specific oxidizing agents such as e.g. iodates, peroxides, ascorbic acid, K-bromate or azodicarbonamide and they are added to dough with the aims of improving the baking performance of flour to achieve a dough with improved stretchability and thus having a desirable strength and stability. The mechanism behind this effect of oxidizing agents is that the flour proteins, in particular gluten contains thiol groups which, when they become oxidized, form disulphide bonds whereby the protein forms a more stable matrix resulting in a better dough quality and improvements of the volume and crumb structure of the baked products.
However, the use of several of the currently available non-specific oxidizing agents is either objected to by consumers or is not permitted by regulatory bodies. Hence it has been attempted to find alternatives to these conventional flour and dough additives, and the prior art has i.a. suggested the use of glucose oxidase and hexose oxidase for this purpose.
Glycerol oxidase is an oxidoreductase which is capable of oxidizing glycerol. Different types of glycerol oxidase have been described in the literature. Some of these glycerol oxidases need co-factors in order to oxidize glycerol (Shuen-Fu et al., 1996. Enzyme Micro. Technol., 18:383-387).
However, glycerol oxidase from Aspergillus japonicus does not require any co-factors in the oxidation of glycerol to glyceraldehyd (T. Uwajima and O. Terada, 1980. Agri. Biol. Chem. 44:2039-2045).
This glycerol oxidase has been characterized by T. Uwajima and O. Terada (Methods in Enzymology, 1982, 89:243-248) and T. Uwajima et al. (Agric. Biol. Chem., 1979, 43:2633-2634), and has a pH optimum at 7.0 and Km and Vmax are 10.4 mM and 935.6 xcexcmol H2O2 minxe2x88x921 respectively using glycerol as substrate. The enzyme is most active on glycerol but also other substrates like dihydroxyacetone, 1,3-propanediol, D-galactose ad D-fructose are oxidized by glycerol oxidase.
Glycerol oxidase not requiring co-factors has also been isolated from Penicillium and characterized by Shuen-Fuh Lin et al. (Enzyme Micro. Technol., 1996, 18:383-387). This enzyme has optimum activity in the pH range from 5.5 to 6.5 at 30xc2x0 C. The enzyme is stable between 20 and 40xc2x0 C. but loses its activity at temperatures above 50xc2x0 C.
Other potential sources for glycerol oxidase according to the invention include different fungal species as disclosed in DE-2817087-A, such as Aspergillus oryzae, Aspergillus parasiticus, Aspergillus flavus, Neurospora crassa, Neurospora sitophila, Neurospora tetrasperma, Penicillium nigricans, Penicillium funiculosum and Penicillium janthinellum. 
Glycerol oxidase isolated from the above natural sources has been used for different applications. Thus, glycerol oxidase from Aspergillus japonicus has been used for glycoaldehyde production from ethylene glycol (Kimiyasu Isobe and Hiroshi Nishise, 1995, Journal of Molecular Catalysis B: Enzymatic, 1:37-43). Glycerol oxidase has also been used in the combination with lipoprotein lipase for the determination of contaminated yolk in egg white (Yioshinori Mie, 1996. Food Research International, 29:81-84). DE-2817087-A and U.S. Pat. No. 4,399,218 disclose the use of glycerol oxidase for the determination of glycerol.
It has now been found that the addition of a glycerol oxidase to a flour dough results in an increased resistance hereof to deformation when the dough is stretched, i.e. this enzyme confers to the dough an increased strength whereby the dough becomes less prone to mechanical deformation. Accordingly, glycerol oxidase is highly useful as a dough conditioning agent in the manufacturing of flour dough based products including not only bread products but also other products made from flour doughs such as noodles and alimentary paste products.
It has also been found that the dough strengthening effect of glycerol oxidase is potentiated significantly when it is combined with a lipase, which in itself does not affect the dough strength. Furthermore, the combined use of glycerol oxidase and lipase results in an improvement of bread quality, in particular in respect of specific volume and crumb homogeneity, which is not a simple additive effect, but reflects a synergistic effect of these two types of enzymes.
Accordingly, the invention relates in a first aspect to a method of improving the rheological properties of a flour dough and the quality of the finished product made from the dough, comprising adding to the dough 10 to 10,000 units of a glycerol oxidase per kg of flour.
In a further aspect there is provided a method of improving the rheological properties of a flour dough and the quality of the finished product made from the dough, comprising adding to the dough a glycerol oxidase and a lipase.
The invention pertains in a still further aspect to dough improving composition comprising a glycerol oxidase and at least one further dough ingredient or dough additive.
In still further aspects, the invention relates to the use of a glycerol oxidase for improving the rheological properties of a flour dough and the quality of the finished product made from the dough and to the use of a glycerol oxidase and a lipase in combination for improving the rheological properties of a flour dough and the quality of the finished product made from the dough.
In one aspect, the present method provides a method of improving the rheological properties of flour doughs.
The expression xe2x80x9crheological propertiesxe2x80x9d as used herein refers particularly to the effects of dough conditioners on dough strength and stability as the most important characteristics of flour doughs. According to American Association of Cereal Chemists (AACC) Method 36-01A the term xe2x80x9cstabilityxe2x80x9d can be defined as xe2x80x9cthe range of dough time over which a positive response is obtained and that property of a rounded dough by which it resists flattening under its own weight over a course of timexe2x80x9d. According to the same method, the term xe2x80x9cresponsexe2x80x9d is defined as xe2x80x9cthe reaction of dough to a known and specific stimulus, substance or set of conditions, usually determined by baking it in comparison with a controlxe2x80x9d
As it is mentioned above, it is generally desirable to improve the baking performance of flour to achieve a dough with improved stretchability and thus having a desirable strength and stability by adding oxidizing agents which cause the formation of protein disulphide bonds whereby the protein forms a more stable matrix resulting in a better dough quality and improvements of the volume and crumb structure of baked products.
Thus, the term xe2x80x9crheological propertiesxe2x80x9d relates to the above physical and chemical phenomena which in combination will determine the performance of flour doughs and thereby also the quality of the resulting products.
The method comprises, as it is mentioned above, the addition of an effective amount of a glycerol oxidase to the dough. It will be understood that the addition can be either to a component of the dough recipe or to the dough resulting from mixing all of the components for the dough. In the present context, xe2x80x9can effective amountxe2x80x9d is used to indicate that the amount is sufficient to confer to the dough and/or the finished product improved characteristics as defined herein. Specifically, such an amount is in the range of 10 to 10,000 units of glycerol oxidase per kg flour.
In one useful embodiment of the method according to the invention, the glycerol oxidase can, as it is described in details herein, be isolated from a bacterial species, a fungal species, a yeast species, an animal cell including a human cell or a plant cell. Examples of glycerol oxidase producing fungal species are species belonging to the genera Aspergillus, Neurospora and Penicillium, such as A. japonicus, A. oryzae, A. parasiticus, A. flavus, Neurospora crassa, N. sitophila, N. tetrasperma, Penicillium nigricans, P. funiculosum and P. janthinellum. 
Glycerol oxidase can be derived as a native enzyme from natural sources such as the above.
It is one objective of the invention to provide improved bakery products. In accordance with the invention, a bakery product dough including a bread dough is prepared by mixing flour with water, a leavening agent such as yeast or a conventional chemical leavening agent, and an effective amount of glycerol oxidase under dough forming conditions. It is, however, within the scope of the invention that further components can be added to the dough mixture.
Typically, such further dough components include conventionally used dough components such as salt, sweetening agents such as sugars, syrups or artificial sweetening agents, lipid substances including shortening, margarine, butter or an animal or vegetable oil, glycerol and one or more dough additives such as emulsifying agents, starch degrading enzymes, cellulose or hemicellulose degrading enzymes, proteases, lipases, non-specific oxidizing agents such as those mentioned above, flavouring agents, lactic acid bacterial cultures, vitamins, minerals, hydrocolloids such as alginates, carrageenans, pectins, vegetable gums including e.g. guar gum and locust bean gum, and dietary fiber substances.
Conventional emulsifying agents used in making flour dough products include as examples monoglycerides, diacetyl tartaric acid esters of mono- and diglycerides of fatty acids, and lecithins e.g. obtained from soya. Among starch degrading enzymes, amylases are particularly useful as dough improving additives. Other useful starch degrading enzymes which may be added to a dough composition include glucoamylases and pullulanases. In the present context, further interesting enzymes are xylanases and oxidoreductases such as glucose oxidase, pyranose oxidase, hexose oxidase, sulfhydryl oxidase, and lipases.
A preferred flour is wheat flour, but doughs comprising flour derived from other cereal species such as from rice, maize, barley, rye and durra are also contemplated.
In accordance with the invention, the dough is prepared by admixing flour, water, the glycerol oxidase and optionally other ingredients and additives. The glycerol oxidase can be added together with any dough ingredient including the water or dough ingredient mixture or with any additive or additive mixture. The dough can be prepared by any conventional dough preparation method common in the baking industry or in any other industry making flour dough based products.
The glycerol oxidase can be added as a liquid preparation or in the form of a dry powder composition either comprising the enzyme as the sole active component or in admixture with one or more other dough ingredients or additive.
The amount of the glycerol oxidase added is an amount which results in the presence in the dough of 10 to 5,000 units (as defined in the following) such as 10 to 2,500 units per kg of flour. In useful embodiments, the amount is in the range of 20 to 1,500 units per kg of flour.
The effect of the glycerol oxidase on the Theological properties of the dough can be measured by standard methods according to the International Association of Cereal Chemistry (ICC) and the American Association of Cereal Chemistry (AACC) including the amylograph method (ICC 126), the farinograph method (AACC 54-21) and the extensigraph method (AACC 54-10). The AACC method 54-10 defines the extensigraph in the following manner: xe2x80x9cthe extensigraph records a load-extension curve for a test piece of dough until it breaks. Characteristics of load-extension curves or extensigrams are used to assess general quality of flour and its responses to improving agentsxe2x80x9d. In effect, the extensigraph method measures the relative strength of a dough. A strong dough exhibits a higher and, in some cases, a longer extensigraph curve than does a weak dough.
In a preferred embodiment of the method according to the invention, the resistance to extension of the dough in terms of the ratio between the resistance to extension (height of curve, B) and the extensibility (length of curve, C), i.e. the B/C ratio as measured by the AACC method 54-10 is increased by at least 10% relative to that of an otherwise similar dough not containing glycerol oxidase. In more preferred embodiments, the resistance to extension is increased by at least 20%, such as at least 50% and in particular by at least 100%.
It has been found that the addition of glycerol oxidase to bakery product doughs results in bakery products such as yeast leavened and chemically leavened products in which the specific volume is increased relative to an otherwise similar bakery product, prepared from a dough not containing glycerol oxidase. In this context, the expression xe2x80x9cspecific volumexe2x80x9d is used to indicate the ratio between volume and weight of the product. It has been found that, in accordance with the above method, the specific volume can be increased significantly such as by at least 10%, preferably by at least 20%, including by at least 30%, preferably by at least 40% and more preferably by at least 50%.
The method according to the invention is highly suitable for improving the rheological properties and quality of the finished products of conventional types of yeast leavened bread products based on wheat flour, such as loaves and rolls. The method is also suitable for improving the rheological properties of doughs containing chemical leavening agents (baking powder) and the quality of products made from such doughs. Such product include as examples sponge cakes and muffins.
In one interesting aspect, the invention is used to improve the Theological properties of doughs intended for noodle products including xe2x80x9cwhite noodlesxe2x80x9d and xe2x80x9cchinese noodlesxe2x80x9d and to improve the textural qualities of the finished noodle products. A typical basic recipe for the manufacturing of noodles comprises the following ingredients: wheat flour 100 parts, salt 0.5 parts and water 33 parts. Furthermore, glycerol is often added to the noodle dough. The noodles are typically prepared by mixing the ingredients in an appropriate mixing apparatus followed by rolling out the noodle dough using an appropriate noodle machine to form the noodle strings which are subsequently air dried.
The quality of the finished noodles is assessed i.a. by their colour, cooking quality and texture. The noodles should cook as quickly as possible, remain firm after cooking and should preferably not loose any solids to the cooking water. On serving the noodles should preferably have a smooth and firm surface not showing stickiness and provide a firm xe2x80x9cbitexe2x80x9d and a good mouthfeel. Furthermore, it is important that the white noodles have a light colour.
Since the appropriateness of wheat flour for providing noodles having the desired textural and eating qualities may vary according to the year and the growth area, it is usual to add noodle improvers to the dough in order to compensate for sub-optimal quality of the flour. Typically, such improvers will comprise dietary fiber substances, vegetable proteins, emulsifiers and hydrocolloids such as e.g. alginates, carrageenans, pectins, vegetable gums including guar gum and locust bean gum, and amylases, and as mentioned above, glycerol.
It is therefore an important aspect of the invention that the glycerol oxidase according to the invention is useful as a noodle improving agent optionally in combination with glycerol and other components currently used to improve the quality of noodles. Thus, it is contemplated that noodles prepared in accordance with the above method will have improved properties with respect to colour, cooking and eating qualities including a firm, elastic and non-sticky texture and consistency.
In a further useful embodiment, the dough which is prepared by the method according to the invention is a dough for preparing an alimentary paste product. Such products which include as examples spaghetti and maccaroni are typically prepared from a dough comprising main ingredients such as flour, eggs or egg powder and/or water. After mixing of the ingredient, the dough is formed to the desired type of paste product and air dried. It is contemplated that the addition of glycerol oxidase to a paste dough, optionally in combination with glycerol, will have a significant improving effect on the extensibility and stability hereof resulting in finished paste product having improved textural and eating qualities.
In a useful embodiment, there is provided a dough improving method wherein at least one further enzyme is added to the dough ingredient, dough additive or the dough. In the present context, suitable enzymes include cellulases, hemicellulases, xylanases, starch degrading enzymes, oxidoreductases and proteases.
In a further aspect, the invention relates to a method of improving the rheological properties of a flour dough and the quality of the finished products made from the dough which comprises that both a glycerol oxidase and a lipase is added to the dough.
It was surprisingly found that the two types of enzymes were capable of interacting with each other under the dough conditions to an extent where the effect on improvement of the dough strength and bread quality by the enzymes was not only additive, but the effect was synergistic.
Thus, with respect to improvement of dough strength it was found that with glycerol oxidase alone, the B/C ratio as measured after 45 minutes of resting was increased by 34%, with lipase alone no effect was observed. However, when combining the two enzymes, the B/C ratio was increased by 54%, i.e. combining the glycerol oxidase with the lipase enhanced the dough strengthening effect of glycerol oxidase by more than 50%. Thus, one objective of combining glycerol oxidase and a lipase is to provide an enhancement of the dough strengthening effect of glycerol oxidase by at least 25% such as at least 50% including at least 75%, determined as described herein.
In relation to improvement of finished product, it was found that the combined addition of glycerol oxidase and a lipase resulted in a substantial synergistic effect in respect to crumb homogeneity as defined herein. Also, with respect to the specific volume of baked product a synergistic effect was found. Thus, for a bread product, the addition of lipase alone typically results in a negligible increase of the specific volume, addition of glycerol oxidase alone in an increase of about 25%, whereas a combined addition of the two enzymes results in an increase of more than 30%.
Further in relation to improvement of the finished product, it was found that the addition of lipase resulted in modification of the glycolipids, monogalactosyl diglyceride and digalactosyl diglyceride present in dough. These components were converted to the more polar components monogalactosyl monoglyceride and digalactosyl monoglyceride. As galactosyl monoglycerides are more surface active components than galactosyl diglycerides it is assumed that galactosyl monoglycerides contributed to the observed improved crumb cell structure and homogeneity. Thus, one objective of using lipase is to hydolyse at least 10% of the galactosyl diglycerides normally present in a flour dough to the corresponding galactosyl monoglycerides, such as at least 50% including at least 100%.
The details of such a method using combined addition of glycerol oxidase and lipase are, apart from the use of a lipase in combination with glycerol oxidase, substantially similar to those described above for a method according to the invention which does not require the addition of a lipase.
When using, in accordance with the invention, a lipase in combination with a glycerol oxidase, the amount of lipase is typically in the range of 10 to 100,000 lipase units (LUS) (as defined in the following) per kg flour including the range of 10 to 20,000 LUS, e.g. 100 to 15,000 LUS such as 500 to 10,000 LUS.
Lipases that are useful in the present invention can be derived from a bacterial species, a fungal species, a yeast species, an animal cell and a plant cell. Whereas the enzyme may be provided by cultivating cultures of such source organisms naturally producing lipase, it may be more convenient and cost-effective to produce it by means of genetically modified cells such as it is described in details in the following examples. In the latter case, the term xe2x80x9cderivedxe2x80x9d may imply that a gene coding for the lipase is isolated from a source organism and inserted into a host cell capable of expressing the gene.
Thus, the enzyme may in a useful embodiment be derived from an Aspergillus species including as examples A. tubigensis, A. oryzae and A. niger. 
Presently preferred lipases include the lipase designated Lipase 3, the production and characteristics of which is described in details in the following examples, or a mutant of this enzyme. In the present context, the term xe2x80x9cmutantxe2x80x9d refers to a lipase having, relative to the wild-type enzyme, an altered amino acid sequence. A further preferred lipase is the lipase found in the commercial product, GRINDAMYL(trademark) EXEL 16.
In a further aspect of the invention there is provided a dough improving composition comprising a glycerol oxidase and at least one further dough ingredient or dough additive.
The further ingredient or additive can be any of the ingredients or additives which are described above. The composition may conveniently be a liquid preparation comprising the glycerol oxidase. However, the composition is conveniently in the form of a dry composition.
The amount of the glycerol oxidase in the composition is in the range of 10 to 10,000 units per kg flour. It will be appreciated that this indication of the amount of enzyme implies that a recommended appropriate amount of the composition will result in the above stated amount in the dough to which it is added. In specific embodiments, the amount of glycerol oxidase is in the range of 10 to 5,000 units such as 10 to 2,500 units per kg of flour. In other useful embodiments, the amount is in the range of 20 to 1,500 units per kg of flour.
In another embodiment, the dough improving composition may further comprises a lipase as defined above and in the amounts as also described above in relation to the method according to the invention.
Optionally, the composition is in the form of a complete dough additive mixture or pre-mixture for making a particular finished product and containing all of the dry ingredients and additives for such a dough. In specific embodiments, the composition is one particularly useful for preparing a baking product or in the making of a noodle product or an alimentary paste product.
In one advantageous embodiment of the above method at least one further enzyme is added to the dough. Suitable examples hereof include a cellulase, a hemicellulase, a xylanase, a starch degrading enzyme, hexose oxidase and a protease.
In a preferred advantageous embodiment, the further added enzyme is a lipase. It has been found that in accordance with the above method, the crumb homogeneity and specific volume of the bakery product can be increased significantly as compared to that of an otherwise similar bakery product prepared from a dough not containing glycerol oxidase, and from a similar bakery product prepared from a dough containing glycerol oxidase.
In a still further aspect, the present invention pertains to the use of a glycerol oxidase and a lipase in combination for improving the rheological properties of a flour dough and the quality of the finished product made from the dough.
In this connection, specific embodiments include use wherein the improvement of the rheological properties of the dough include that the resistance to extension of the dough in terms of the ratio between resistance to extension (height of curve, B) and the extensibility (length of curve, C), i.e. the B/C ratio, as measured by the AACC method 54-10 is increased by at least 10% relative to that of an otherwise similar dough that does not contain glycerol oxidase and use wherein the improvement of the quality of the finished product made from the dough is that the average pore diameter of the crumb of the bread made from the dough is reduced by at least 10%, relative to a bread which is made from a bread dough without addition of the lipase.
In a further embodiment, the use according to the invention, implies that the improvement of the quality of the finished product made from the dough consists in that the pore homogeneity of the crumb of the bread made from the dough is incresed by at least 5%, relative to a bread which is made from a bread dough without addition of the lipase. The pore homogeneity of bread is conveniently measured by means of an image analyzer composed of a standard CCD-video camera, a video digitiser and a personal computer with WinGrain software. Using such an analyzer, the results of pore diameter in mm and pore homogeneity can be calculated as an average of measurements from 10 slices of bread. The pore homogeneity is expressed in % of pores that are larger than 0.5 times the average of pore diameter and smaller than 2 times the average diameter.
In a further embodiment, the use relates to improvement of the rheological characteristics of the dough including that the gluten index (as defined hereinbelow) in the dough is increased by at least 5%, relative to a dough without addition of a lipase, the gluten index is determined by means of a Glutomatic 2200 apparatus.